MULTIPLEXING HOMOGENEOUS CELL-BASED ASSAYS
ABIGAIL FARFAN, M.A., THOMAS YEAGER, PH.D., RICH MORAVEC, B.S., AND ANDREW NILES, M.S., PROMEGA CORPORATION
Researchers often desire more than one type of data from a cell sample in order to gain a more complete understanding of the
biological processes that they are investigating. Here we provide brief protocols for performing a combination of cell viability, cytotoxicity, apoptosis or reporter assays on a single sample.
Introduction
In the area of apoptosis Promega offers a range of fluorescent
and luminescent methods to measure caspase-3/7, caspase-8
and caspase-9 activities that can be used in parallel, as well as
in multiplexing formats, with luminescent or fluorescent cell viability, cytotoxicity and reporter assays. This approach can provide a more complete understanding of how a compound treatment affects cell viability and/or the mechanism of cell death.
The high-throughput demands of modern biomedical research
have affected everything from nucleic acid purification technologies to cell-based assays including reporter analysis. The
latest generation of Promega cell-based assays include luminescent and fluorescent chemistries to measure markers of cell
viability, cytotoxicity, and apoptosis as well as to perform
reporter analysis. Using these tools, researchers can investigate
how cells respond to growth factors, cytokines, hormones, mitogens, radiation, effectors and other signaling ligands. In drug
discovery, these assays provide efficient means for testing drug
candidates for toxicity or efficacy before investing in animal
research and clinical trials.
The tables that follow provide basic guidelines for multiplexing
cell-based assays and are intended as starting points. As with
any homogeneous assay, multiplexing assays will require
researchers to optimize the assays for any specific experimental
system. We strongly recommend running appropriate controls,
including performing each assay individually on the samples.
Additional background, optimization and control information for
each assay is provided in the accompanying technical literature, listed at the end of this article. For a complete review to
help you choose the right cell-based assays to address your
experimental questions, please see references 1, 2 or 4.
However, researchers often need more than one type of data
from a sample, so the ability to multiplex, to analyze more than
one parameter from a single sample, is desirable. Promega cellbased assays are homogeneous, that is they can be performed
directly in cell culture wells without removing medium or washing cells. This homogeneous format allows researchers to multiplex assays (Table 1). Multiplexing more than one assay from
the same culture well can provide internal controls and eliminate the need to repeat work. For example, a researcher can
choose to perform a fluorescent assay to measure cytotoxicity
or viability while next performing a luminescent caspase activity
assay or reporter assay on the same sample(a). Multiplexing
more than one assay from the same culture well can also save
time, cell sample, cell culture reagent and rare or expensive
test compounds.
Multiplexing Cell Viability, Cytotoxicity and
Apoptosis Assays
The CellTiter-Blue® Cell Viability Assay determines the number
of viable cells in culture using resazurin to characterize the
reducing potential of the cells. The reduction of resazurin to
resorufin is proportional to the number of metabolically active,
cells present. The CellTiter-Blue® Assay can be multiplexed with
either the Caspase-Glo™ 3/7 Assay(b,c) or the Apo-ONE®
Homogeneous Caspase-3/7 Assay (Table 2).
Table 1. Summary of Promega Cell-Based Assays for Multiplexing
Detection Method
Measures
Multiplex with:
CellTiter-Glo® Luminescent Cell Viability Assay
Luminescent
ATP,
EnduRen™ Live Cell Substrate (Renilla reporter)
viability
CytoTox-ONE™ Homogeneous Membrane Integrity Assay (fluorescent)
CellTiter-Blue® Cell Viability Assay
Fluorescent
Reducing Potential,
Caspase-Glo™ 3/7 Assay (luminescent)
viability
Apo-ONE® Homogeneous Caspase 3/7 Assay (fluorescent)
CytoTox-ONE™ Homogeneous Membrane
Fluorescent
LDH Release,
Caspase-Glo™ 3/7 Assay (luminescent)
cytotoxicity
Apo-ONE® Homogeneous Caspase 3/7 Assay (fluorescent)
Caspase-Glo™ 3/7 Assay
Luminescent
Caspase-3/7 Activity
CellTiter-Blue® Cell Viability Assay (fluorescent)
Caspase-Glo™ 8 or Caspase-Glo™ 9 Assay
Luminescent
Caspase-8 or -9 Activity Apo-ONE® Homogeneous Caspase 3/7 Assay (fluorescent)
Apo-ONE® Homogeneous Caspase 3/7 Assay
Fluorescent
Caspase 3/7 Activity
Integrity Assay
CytoTox-ONE™ Homogeneous Membrane Integrity Assay (fluorescent)
Caspase-Glo® 8 or Caspase-Glo® 9 Assays (luminescent)
CellTiter-Blue® Cell Viability Assay (fluorescent)
CytoTox-ONE™ Homogeneous Membrane Integrity Assay (fluorescent)
EnduRen™ Live Cell Substrate (Renilla reporter)
EnduRen™ Live Cell Substrate
www.promega.com
Luminescent
Renilla Reporter
CellTiter-Glo® Luminescent Cell Viability Assay (luminescent)
Gene Activity
Apo-ONE® Homogeneous Caspase 3/7 Assay (fluorescent)
15
CELL NOTES ISSUE 10 2004
CELL-BASED ASSAYS
Assay
Multiplexing Cell-Based Assays
6
1
2
3
4
5
6
7
8
9
Duration of Drug Exposure (Hours)
10
Figure 1. Multiplexing luminescent caspase-8 and fluorescent caspase
3/7 assays. Jurkat cells were seeded at 25,000 cells/well. 50µl of
rTRAIL (Chemicon, 100ng/ml final) or a vehicle control (RPMI 1640
with 10% FBS) was added to replicate wells every hour for 10 hours.
Caspase-Glo™ 8 Reagent was prepared by combining the assay buffer
with the substrate. A fluorescent caspase-3/7 substrate [(Z-DEVD)2R110] was mixed into the Caspase-Glo™ Reagent at a final
concentration of 50µM. The combined Reagent/substrate was added
in 100µl volumes, incubated 60 minutes, and then luminescence and
fluorescence were measured.
The CytoTox-ONE™ Homogeneous Membrane Integrity
Assay(a) measures the release of lactate dehydrogenase (LDH)
into the surrounding medium by cells that have lost membrane integrity. The assay estimates the number of nonviable
cells present in a mixed population of living and dead cells.
The CytoTox-ONE™ Reagent does not damage living cells and
can be performed directly in cell culture. Tables 2 and 3
describe how the CytoTox-ONE™ Assay can be multiplexed
with either the Caspase-Glo™ 3/7 Assay, the CellTiter-Glo®
Luminescent Cell Viability Assay, or the Apo-ONE® Assay.
CellTiter-Glo® Luminescent Cell Viability Assay determines the
number of viable cells in culture based on quantitation of ATP
present, an indicator of metabolically active cells. It can be
multiplexed with either EnduRen™ Live Cell Substrate (Table
4 and Figure 2) or CytoTox-ONE™ Homogeneous Membrane
Integrity Assay (Tables 2 and 3).
Multiplexing Apoptosis Assays
The Caspase-Glo™ Assays measure caspase activities using
a luminogenic caspase substrate and a proprietary stabilized
luciferase in a reagent optimized for specific caspase activity,
luciferase activity and cell lysis. Adding the single
Caspase-Glo™ Reagent in an "add-mix-measure" format
results in cell lysis, followed by caspase cleavage of the substrate. This cleavage liberates free aminoluciferin, which is
consumed by the luciferase, generating a "glow-type" luminescent signal. The signal is proportional to caspase activity present. Currently Caspase-Glo™ Assays are available to measure
caspase-3/7, caspase-8 or caspase-9 activity. These assays
can be multiplexed with cell viability and cytotoxicity assays
(Table 2). The Caspase-Glo™ 8 or 9 Assays can be multiplexed with the Apo-ONE® Homogeneous Caspase-3/7 Assay
to provide a more complete picture of apoptotic signaling
(Figure 1,Table 2).
The Apo-ONE® Homogeneous Caspase-3/7 Assay measures
active caspase-3 and -7 by including a profluorescent casCELL NOTES ISSUE 10 2004
8
6
1,000
4
500
Renilla RLU x 103
10
10
1,500
2
0
0
rTRAIL (µg/ml)
4753MA
14
12
2,000
0. 0
0
0. 08
0
0. 16
0
0. 31
0
0. 63
12
0. 5
25
0.
5
1.
0
2.
0
4.
0
18
CellTiter-Glo® RLU x 103
22
14
2,500
Caspase-8 RLU
1,200
1,100
1,000
900
800
700
600
500
4754MA
Caspase-3/7 RFU x 103
CELL-BASED ASSAYS
26
0
CellTiter-Glo® Assay
EnduRen™ Assay
Vehicle Caspase-8
Vehicle Caspase-3/7
T RA IL Caspase-8
T RA IL Caspase-3/7
Figure 2. Multiplex of Renilla reporter assay with a luminescent cell
viability assay. HeLa cells stably expressing a synthetic Renilla
luciferase gene were plated at 10, 000 cells/well in a 96-well plate.
EnduRen™ Live Cell Substrate (Cat.# E6482) was added to all the
wells at a 1:1,000 dilution in DMEM + 10% FBS. The TRAIL protein
(CalBiochem Cat.# 616375) was added to the indicated wells starting
at 4µg/ml with subsequent twofold serial dilutions. Cells were
incubated for 16 hours and assayed. Renilla expression was
measured using a Veritas™ Microplate Luminometer (Cat.# E6521)
immediately after incubation. CellTiter-Glo® Reagent was then added
at a volume of 1:1 to each well and luminescence read on the
Veritas™ Microplate Luminometer.
pase-3/7 consensus substrate and an optimized bifunctional
cell lysis/activity buffer. The buffer lyses cultured mammalian
cells and supports optimal caspase-3/7 enzymatic activity. The
substrate and buffer are combined to make the Apo-ONE®
Caspase-3/7 Reagent that is added directly to samples. Upon
cleavage on the C-terminal side of the aspartate residue in the
DEVD peptide substrate sequence by caspase-3/7 enzymes,
the rhodamine 110 becomes fluorescent when excited at a
wavelength of 498nm. The amount of fluorescent product generated is representative of the amount of active caspase-3/7
present in the sample.
Multiplexing Reporter Assays with Cell Viability
and Apoptosis Assays
The EnduRen™ Live Cell Substrate(c,d,e) is a non-destructive
assay that allows repeated Renilla reporter gene activity
measurements over a period of time or allows users to multiplex with cell viability or apoptosis assays and potentially other
lytic assays. Multiplexing a reporter assay with a cell viability
assay allows researchers to normalize reporter assay data with
respect to cell viability and reduce relative error of results
(Figure 2, Table 4). EnduRen™ Live Cell Substrate also provides researchers with a tool to investigate caspase activity
and reporter activity in the same sample (Table 4).
Summary
Multiplexing is an efficient way to normalize cell-based data to
understand complex cellular processes. The range of homogeneous cell-based assays available from Promega gives
researchers the flexibility and tools they need to dissect
complex cellular processes. ■
16
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Multiplexing Cell-Based Assays
Table 2. Sequential Multiplexing of Promega Homogeneous Cell Viability and Apoptosis Assays (same well).
Assay Goal: Determine cell viability and the mechanism of death. CellTiter-Blue® Assay (viability, fluorescent) and Apo-ONE® Assay (caspase-3/7, fluorescent)
1.
Culture and treat cells with drug of interest in 100µl of medium in a 96-well plate (black or white).
2.
Add 20µl/well CellTiter-Blue® Reagent during the final 1–2 hours of drug treatment and incubate at 37°C.
3.
Record fluorescence (560Ex/590Em) as described in Technical Bulletin #TB317 to measure cell viability.
4.
Add an equal volume (120µl) Apo-ONE® Homogeneous Caspase-3/7 Reagent and incubate for 1–4 hours at room temperature.
5.
Record fluorescence (485Ex//527Em) as described in Technical Bulletin #TB295 to indicate caspase activity as a marker of apoptosis.
Assay Goal: Determine cell viability and the mechanism of death. CellTiter-Blue® Assay (viability, fluorescent) and Caspase-Glo™ 3/7 Assay (luminescent)
1.
Culture and treat cells with drug of interest in 100µl of medium in a 96-well plate (black or white).
2.
Add 20µl/well of CellTiter-Blue® Reagent (diluted 1:4 with Dulbecco’s PBS) during the final 1–2 hours of drug treatment and incubate at 37°C.
3.
Record fluorescence (560Ex/590Em) as described in Technical Bulletin #TB317 to measure cell viability.
4.
Add an equal volume (120µl) of Caspase-Glo™ 3/7 Reagent to each well. and incubate 1 hour at room temperature to achieve luciferase steady state.
5.
Record luminescence as described in Technical Bulletin #TB323 to indicate caspase activity as a marker of apoptosis.
Note: Ensure that all of the wells change to an even pink color after incubating with Caspase-Glo™ Reagent. If all of the wells contain the same pink color when luminescence is recorded, the light is quenched evenly throughout the sample, regardless of the initial CellTiter-Blue® activity.
Assay Goal: Determine the mechanism of compound cytotoxicity. CytoTox-ONE™ Assay (LDH release, fluorescent) and Caspase-Glo™ 3/7 Assay (luminescent)
1.
Culture and treat cells with drug of interest in 100µl of medium in a 96-well plate (black or white).
2.
Reconstitute CytoTox-ONE™ Substrate at 2X concentration and add 25µl/well.
3.
Shake while incubating 10 minutes at room temperature. Record fluorescence (560Ex/590Em) as described in Technical Bulletin #TB306 to measure necrotic cells.
4.
Add an equal volume (125µl) of Caspase-Glo™ Reagent to each well.
5.
Incubate for 1 hour at room temperature to achieve luminescence steady state. Record luminescence as described in Technical Bulletin #TB323.
Note: Ensure that all of the wells change to an even pink color after incubating with Caspase-Glo™ Reagent. If all of the wells contain the same pink color when luminescence is recorded, the light is quenched evenly throughout the sample, regardless of the initial CytoTox-ONE™ activity.
Assay Goal: Determine cytotoxicity and cell viability. CytoTox-ONE™ Assay (LDH release, fluorescent) and CellTiter-Glo® Assay (cell viability, ATP)
1.
Culture and treat cells with drug of interest in 75µl of medium in a 96-well plate (black or white).
2.
Reconstitute CytoTox-ONE™ Substrate at 1X concentration, and add 50µl/well.
3.
Shake gently and incubate 10 minutes at room temeprature. Record fluorescence (560Ex/590Em) as described in Technical Bulletin #TB306.
4.
Reconstitute the CellTiter-Glo® Substrate and add 20mM DTT. Add an equal volume (125µl) to each well.
5.
Shake gently and incubate for 1 hour at room temperature. Record luminescence as described in Technical Bulletin #TB288.
Note: Ensure that all of the wells change to an even pink color after incubating with CellTiter-Glo® Reagent. If all of the wells contain the same pink color when luminescence is recorded, the light is quenched evenly throughout the sample, regardless of the initial CytoTox-ONE™ activity.
Assay Goal: Differentiate caspase activities. Caspase-Glo™ 8 or 9 Assay (luminescent) and Apo-ONE® Assay (caspase-3/7 activity, fluorescent)
1.
Culture and treat cells with drug of interest in 100µl of medium in a 96-well plate (black or white).
2.
Prepare the Caspase-Glo™ 8 or 9 Assay Reagent. Thaw the Apo-ONE® Substrate and add it to the Caspase-Glo™ 8 or 9 Reagent at a dilution of 1:200
(50µl/10ml of Caspase-Glo™ Reagent). The Apo-ONE® Buffer will not be used in this assay. Add an equal volume (100µl) to each well.
3.
Incubate 1 hour at room temperature. Record luminescence as described in Technical Bulletin #TB332 or #TB333 to indicate caspase-8 or -9 activity.
4.
Record Apo-ONE® Homogeneous Caspase-3/7 Assay fluorescence (485Ex/527Em) as described in Technical Bulletin #TB295 to indicate caspase-3 activity.
References
1. Riss, T. et al. (2003) Cell Notes 6, 6–12.
2. Niles, A. et al. (2004) Cell Notes 9, 11–14.
3. Riss, T. and Moravec, R. (2003) Promega Notes 83, 10–13.
4. Riss, T. and Moravec, R. (2004) Assay Drug Dev. Techol. 2, 51–62.
Caspase-Glo™ 3/7 Assay Technical Bulletin #TB323
(www.promega.com/tbs/tb323/tb323.html)
Caspase-Glo™ 8 Assay Technical Bulletin #TB332
(www.promega.com/tbs/tb332/tb332.html)
CellTiter-Glo ®
Luminescent Cell Viability Assay
Technical Bulletin #TB288
(www.promega.com/tbs/tb288/tb288.html)
Caspase-Glo™ 9 Assay Technical Bulletin #TB333
(www.promega.com/tbs/tb333/tb333.html)
CellTiter-Blue ® Cell Viability Assay Technical Bulletin #TB317
(www.promega.com/tbs/tb317/tb317.html)
EnduRen™ Live Cell Substrate Technical Manual #TM244
(www.promega.com/tbs/tm244/tm244.html)
CytoTox-ONE ™ Homogeneous Membrane Integrity Assay
Technical Bulletin #TB306
(www.promega.com/tbs/tb306/tb306.html)
www.promega.com
17
CELL NOTES ISSUE 10 2004
CELL-BASED ASSAYS
Protocols
Apo-ONE ® Homogeneous Caspase-3/7 Assay Technical
Bulletin #TB295
(www.promega.com/tbs/tb295/tb295.html)
Multiplexing Cell-Based Assays
CELL-BASED ASSAYS
Table 3. Multiplexing Promega Homogeneous Cell Viability and Apoptosis Assays (medium and cells separately).
Assay Goal: Determine cytotoxicity and caspase-3/7 activity. CytoTox-ONE™ Assay (LDH release, fluorescent) and Apo-ONE® Assay (caspase-3/7 activity, fluorescent)
1.
Culture and treat cells with drug of interest in 100µl of medium in a 96-well plate (black or white).
2.
Transfer supernatant to a new plate and add CytoTox-ONE™ Reagent to measure LDH release indicating necrotic cells.
3.
Incubate and record flourescence (560Ex/590Em) as described in Technical Bulletin #TB306.
4.
Add Apo-ONE® Homogeneous Caspase-3/7 Reagent to the cells in the original plate and incubate for 1–4 hours at room temperature.
5.
Record Apo-ONE® Assay fluorescence (485Ex/527Em) as described in Technical Bulletin #TB295.
Assay Goal: Determine cytotoxicity and cell viability. CytoTox-ONE™ Assay (LDH release, fluorescent) and CellTiter-Glo® Assay (viability, luminescent)
1.
Culture and treat cells with drug of interest in 100µl of medium in a 96-well plate (black or white).
2.
Transfer supernatant to a new plate and add CytoTox-ONE™ Reagent.
3.
Incubate and record flourescence (560Ex/590Em) as described in Technical Bulletin #TB306.
4.
Add CellTiter-Glo® Reagent to cells in original sample plate. Incubate 10 minutes and record luminescence as described in Technical Bulletin #TB288.
Table 4. Multiplexing Promega Cell Viability and Reporter Assays
Assay Goal: Normalize reporter gene signal with respect to cell viability. EnduRen™ Live Cell Substrate (Renilla luciferase, luminescent) and CellTiter-Glo® Assay
(viability, luminescent)
1.
Culture and treat cells with the drug of interest in 90µl of medium in a 96-well plate.
2.
Dilute the EnduRen™ Live Cell Substrate as directed in Technical Manual #TM244. Add 10µl/well of EnduRen™ Substrate (60µM) and incubate for an
additional 2 hours at 37°C, 5% CO2. You may add the EnduRen™ Substrate before or after experimental treatment depending on cell tolerance to it.
3.
4.
Record luminescence to indicate reporter activity.
Add an equal volume of CellTiter-Glo® Reagent (100µl/well), mix for 2 minutes on an orbital shaker to induce cell lysis, and incubate and additional 10
minutes at room temperature to stabilize luminescent signal.
5.
Record luminescence as described in Technical Bulletin #TB288 to indicate cell viability.
Assay Goal: Determine gene regulation and apoptosis involvement. EnduRen™ Live Cell Substrate (Renilla luciferase) and Apo-ONE® Assay (caspase-3/7 activity)
1.
Culture and treat cells with the drug of interest in 90µl of medium in a 96-well plate.
2.
Dilute the EnduRen™ Live Cell Substrate as directed in Technical Manual #TM244. Add 10µl/well of EnduRen™ Substrate (60µM) and incubate for an
additional 2 hours at 37°C, 5% CO2. You may add the EnduRen™ Substrate before or after experimental treatment depending on cell tolerance to it.
3.
Record luminescence.
4.
Add an equal volume of Apo-ONE® Reagent (100µl/well) and incubate for 1 hour at room temperature.
5.
Record fluorescence (485Ex/527Em) as described in Technical Bulletin #TB295.
Ordering Information
(a)Patent
Product
Size
Cat.#
Assay(b,c)*
100ml
G8202
Caspase-Glo™ 8 Assay(b,c)*
100ml
G8212
Caspase-Glo™ 3/7 Assay(b,c)*
100ml
G8092
Caspase-Glo™ 8
Pending.
Pat. No. 6,602,677, Australian Pat. No. 754312 and other patents pending.
(c)The method of recombinant expression of Coleoptera luciferase is covered by U.S. Pat. Nos.
5,583,024, 5,674,713 and 5,700,673.
(d)Certain applications of this product may require licenses from others.
(e)This product does not convey a license to use recombinant Renilla luciferase under U.S. Pat.
Nos. 5,292,658, 5,418,155 and related patents. Promega sells licensed Renilla luciferase
vectors, which may be used in conjunction with this product.
(b)U.S.
Apo-ONE® Homogeneous Caspase-3/7 Assay
100ml
G7791
10 × 10ml
G7571
Apo-ONE, CellTiter-Blue and CellTiter-Glo are trademarks of Promega Corporation and are
registered with the U.S. Patent and Trademark Office. Caspase-Glo, CytoTox-ONE and EnduRen
are trademarks of Promega Corporation.
20ml
G8080
Veritas is a trademark of Turner BioSystems, Inc.
CytoTox-ONE™ Homogeneous
Membrane Integrity Assay(a)
1,000–4,000 assays
G7891
CytoTox-ONE™ Homogeneous
Membrane Integrity Assay, HTP(a)
1,000–4,000 assays
G7892
0.34ng
E6482
CellTiter-Glo® Luminescent Cell Viability Assay(b,c)
CellTiter-Blue® Cell Viability Assay
EnduRen™ Live Cell Substrate*(a,d,e)
Available in additional sizes. *For Laboratory Use.
CELL NOTES ISSUE 10 2004
18
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